Heart failure is defined as the inability of the cardiac pump to move blood as needed to provide for the metabolic needs of body tissue. Decreases in pumping ability arise most often from loss or damage of myocardial tissue. As a result, ventricular emptying is suppressed which leads to an increase in ventricular filling pressure and ventricular wall stress, and to a decrease in cardiac output. As a physiological response to the decrease in cardiac output, numerous neuroendocrine reflexes are activated which cause systemic vasoconstriction, sympathetic stimulation of the heart and fluid retention. Although these reflex responses tend to enhance cardiac output initially, they are detrimental in the long term. The resulting increases in peripheral resistance increase the afterload on the heart and the increases in blood volume further increase ventricular filling pressure. These changes, together with the increased sympathetic stimulation of the heart, lead to further and often decompensating demands on the remaining functional myocardium.
Congestive heart failure, which is a common end point for many cardiovascular disorders, results when the heart is unable to adequately perfuse the peripheral tissues. According to recent estimates, there are about 4 million people in the United States diagnosed with this disease, and more than 50% of these cases are fatal within 5 years of diagnosis [Taylor, M.D. et al., Annual Reports in Med, Chem. 22, 85-94 (1987)].
Current therapy for heart failure, including congestive heart failure, focuses on increasing cardiac output without causing undue demands on the myocardium. To achieve these ends, various combinations of diuretics, vasodilators and inotropic agents are used to decrease blood volume, to decrease peripheral resistance, and to increase force of cardiac contraction. Current therapy therefore depends on balancing the effects of multiple drugs to achieve the clinical needs of individual patients, and is plagued by adverse reactions to the drugs used.
For example, diuretics decrease plasma concentrations of potassium and magnesium and increase the incidence of arrhythmias in patients receiving digitalis. Diuretics can potentiate the circulatory effects of nitrates through volume depletion and lead to decreases in filling pressure of the heart, cardiac output and systemic arterial pressure.
Alpha adrenergic antagonists can lead to marked falls in systemic arterial pressure that compromise coronary perfusion.
Angiotensin converting enzyme inhibitors can have similar effects on arterial pressure and additionally lead to excessive increases in plasma concentrations of potassium.
Drugs that lead to positive inotropy, such as digitalis and beta adrenergic antagonists, have the potential to provoke arrhythmias. In addition, digitalis has a narrow therapeutic index and the catecholamine analogs all tend to loose their effectiveness rapidly, due to receptor downregulation.
Thus there is a need for therapeutic agents that lead to physiologically integrated responses of arterial and venous vasodilation and cardiac inotropy, and are devoid of the disadvantages of the currently used therapeutic agents.
Similarly, cardiac arrhythmias affect a large patient population. The impulse setting the heart rate is formed in the sinoatrial node (SA node, sinus node), which is, therefore, also referred to as the primary pacemaker of the heart. Spontaneous action potential formation can also be demonstrated in specialized atrial cells, in some regions of the atrioventricular (AV) junction, and in His-Purkinje fibers. These areas function as subsidiary pacemakers. In the normal heart, transmission of impulses arising in the SA node to the ventricle occurs exclusively via the atrioventricular node (AV node). Although the normal heart rate may vary significantly, depending on many factors, including age, sex, physical activity, the normal sinus rhythm in humans is arbitrarily defined as impulse formation beginning in the sinus node at frequencies between about 60 and 100 beats/minute.
Sinus bradycardia in human patients is defined as a condition when the SA node of an adult discharges at a rate less than about 60 beats/minute. Although the treatment of asymptomatic bradycardias is usually not necessary, treatment is indicated in a variety of circumstances, such as if the cardiac output is inadequate or if arrhythmias are associated with the slow heart rate. Treatment may also be indicated if symptomatic bradycardia is likely to occur, such as after some forms of cardiac surgery, during administration of certain drugs that might inappropriately slow the heart rate, and prior to pacemaker implantation in patients with symptomatic bradycardia.
The current pharmacological approaches to increasing heart rate are usually based on the administration of atropine, or, in the absence of myocardial ischemia, isoproterenol. Ephedrine, epinephrine, hydralazine or theophylline may also be useful in managing some patients with symptomatic bradycardia. All these drugs are accompanied by more or less serious side-effects, and need to be administered with great caution so as not to produce too rapid a heart rate. The effect of atropine is relatively short-lived, the drug is relatively inefficient in a large number of patients, and on occasion can lead to uncontrolled increases in heart rate. Isoproterenol is efficacious if the SA node is functioning appropriately and the conduction to the AV node is intact, but also can lead to ventricular arrhythmias. [See "Heart Disease." A Text of Cardiovascular Medicine, E. Braunwald, ed., 3rd edition, 1988, W.B. Saunders Company, Chapters 22 and 23; Cardiology: Fundamentals and Practice, Brandenburg, R.O. et al., eds. Year Book Medical Publ. Inc., Chicago, London, chapter 29.)
Accordingly, there is a need for therapeutic agents that can be safely administered to titrate the heart rate to a predictable level, and that do not increase ventricular irritability or lower the threshold for ventricular fibrillation. An agent of this nature could, for example, be useful in the treatment of transient bradycardia following an inferior myocardial infarct, to stabilize patients which require the emergent implantation of a temporary pacemaker, and in the management of bradycardia due to overmedication, e.g. with digitalis, S-blockers or calcium antagonists.
Mature human relaxin is a disulfide bridged polypeptide hormone of approximately 6000 daltons, which is known to show a marked increase in concentration during pregnancy in many species, and is known to be responsible for remodelling the reproductive tract before parturition, thus facilitating the birth process.
Relaxin was discovered by F. L. Hisaw [Proc. Soc. Exp. Biol. Med. 23,661 (1962)] and received its name from Fevold et al. [J. Am. Chem. Soc. 52, 3340 (1930)] who obtained a crude aqueous extract of this hormone from sow corpora lutea. A multitude of observations with crude relaxin preparations led to the view that relaxin probably plays an important role during pregnancy and parturition.
Between 1974 and 1981, highly purified relaxin was isolated from the ovaries of pregnant pigs [Sherwood and O'Byrne Arch. Biochem. Biophys. 160, 185 (1974)], rats [Sherwood, O.D., Endocrinology 104, 886 (1979)], and sharks [Reinig et al., Endocrinology 109, 537 (1981)]. More recently, highly purified relaxin was isolated from the placentas of horses [Stewart, D. R. and Papkoff, Endocrinology 119, 1093 (1986)] and rabbits [Eldridge, R. K. and Fields, P. A., in Biology of Relaxin and its Role in the Human M. Bigazzi et al., eds. 389-391, Excerpta Medica, Amsterdam (1983)]. Partially purified relaxin was obtained from cow and human corpora lutea (CL), placentas, and decidua. In the human, relaxin is known to exist in most abundance in the corpora lutea of pregnancy, however, relaxin has also been detected in non-pregnant female as well as in the male (seminal fluid) [Bryant-Greenwood, G. D., Endocrine Reviews 3, 62-90 (1982) and Weisse, G., Ann. Rev. Physiol. 46, 43-52 (1984)].
The availability of purified relaxin has enabled the amino acid sequence determination of relaxin from pig [James et al., Nature 267, 544 (1977); Schwabe et al., Biophys. Res. Commun. 75, 503 (1977)], rat [John et al., Endocrinology 108, 726 (1981)] and shark [Schwabe et al., Ann. N.Y. Acad. Sci. 380, 6 (1982)].
Efforts have been made to purify relaxin from human corpora lutea, placentas and decidua but none of the human relaxin preparations were demonstrated to be highly purified.
Recombinant techniques have first been applied to the isolation of cDNA clones for rat and porcine relaxins [Hudson et al., Nature 291, 127 (1981); Haley et al., DNA 1, 155 (1982)]. Two human gene forms have been identified by genomic cloning using probes from the porcine relaxin gene [Hudson et al., Nature 301, 628 (1983); Hudson et al., EMBO J. 3, 2333 (1984); and U.S. Pat. Nos. 4,758,516 (issued Jul. 19, 1988) and 4,871,670 (issued Oct. 3, 1989)], although only one of these gene forms (termed H2) has been found to be transcribed in corpora lutea. It is unclear whether the other gene is expressed at another tissue site, or whether it represents a pseudo-gene. The fact that H2 relaxin is synthesized and expressed in the ovary suggests that this is the sequence that is directly involved in the physiology of pregnancy.
Relaxin consists of two peptide chains, referred to as A and B, joined by disulfide bonds with an intra-chain disulfide loop in the A-chain in a manner analogous to that of insulin. The two human relaxin genes show considerable nucleotide and amino acid sequence homology to each other, however, there are some notable regions of sequence divergence, particularly in the amino-terminal region of both A- and B-chains.
The structure of relaxin has apparently diverged considerably among species during evolution. Only 40% to 48% amino acid sequence homology exists among porcine, rat, shark, and human relaxins.
Similar to all species examined, the primary translation product of H2 relaxin is a preprorelaxin consisting of a 25 amino acid signal sequence followed by a B chain of about 29-33 amino acids, a connecting peptide of 104-107 amino acids (C peptide), and an A chain of 24 amino acids. During the biosynthesis of relaxin, the signal peptide is removed rapidly as the nascent peptide chain is translocated across the endoplasmic reticulum. The further processing of the prohormone obtained into relaxin, and especially the function of the C peptide in relaxin is not entirely understood. One function presumably is to direct the folding of the precursors so that the correct disulfide bonds are formed between the B and A chains.
A concise review of the knowledge about relaxin as of 1988 was provided by Sherwood, D. in The Physiology of Reproduction Chapter 16 "Relaxin" Knobil E. and Neill, J. et al., (eds) Raven Press, Ltd., New York pp. 585-673 (1988).
It is known that relaxin increases in peripheral plasma 7-10 days after the midcycle surge of luteinizing hormone and continues to rise if conception has occurred, obtaining levels of over 800 pg/ml by three weeks [Stewart, D.R. et al., J. Clin. Endo. Metab. 70, 1771-1773 (1990)]. During pregnancy, serum concentrations of relaxin, as measured by a homologous radioimmunoassay, is highest by about the 10th week, obtaining levels of about 500 pg/ml for the remainder of pregnancy [Bell, R. J. et al., Obstet. Gynecol. 69, 585-589 (1987)].
In view of the above and similar physiological findings, relaxin has been consistently associated with the condition of pregnancy, and most of its known utilities are associated with this condition.
H2 relaxin has been described to remodel the reproductive tract to facilitate birth process, including ripening of the cervix, thickening of the endometrium of the pregnant uterus as well as increased vascularization to this area, and an effect on collagen synthesis. H2 relaxin has also been associated with lactation, and some reports indicate that relaxin has a growth-promoting effect on mammary tissue [Wright, L. C. and Anderson, R. R., Adv. Exp. Med. Biol. 143, 341 (1982)].
It has been observed that Raynaud's lesions completely disappear during early pregnancy. Clinical studies, which derived from this observation, have shown that an ovarian derived porcine relaxin was beneficial in healing Raynaud's lesions arising from obliterative peripheral arterial disease [Casten, G. C. and Boucek, R. J., J. Am. Med. Assoc. 166, 319-324 (1958); Casten, G. C., et al., Angiology 11, 404-414 (1960) ].
There are indications that relaxin might be present in the male reproductive tract. Human seminal plasma was reported to contain relaxin bioactivity [Weiss et al., Am. J. Obstet. Gynecol. 154,749 (1986)], and relaxin is believed to enhance the mobility of human spermatozoa.
Given the effect of relaxin on the connective tissue, it has been suggested that relaxin may improve skin elasticity.
It has been observed that in pregnant women heart rate increases by 2 weeks after conception, showing an elevation of about 7 beats/min, and continues to rise, obtaining elevations of about 10 beats/min by the 10th week [Clapp, J. F., Am. J. Obstet. Gynecol. 152,659-660 (1985)]. This change in early pregnancy is coincident with the first elevation of circulating relaxin in pregnant women [Stewart, D. R. et al., Supra].
Similarly, cardiac output increases and total peripheral resistance decreases by three weeks after conception, showing changes of +0.52 liter/min and -113 dyn.s.sup.31 1.cm.sup.-1, respectively. Cardiac output continues to increase and total peripheral resistance continues to decrease, reaching +2.21 1/min and -433 dyn.s.sup.-1.cm.sup.-5, respectively at between 10 and 14 weeks before leveling off near these values [Robson, S.C. et al., Am. J. Physiol. 256, H160-H1065 (1989)].
There are sporadic reports on the effect of relaxin administration on blood vessels, and blood pressure under special circumstances, without any written indication that the reported observations could have any potential therapeutic implications.
Local application of porcine relaxin to the rat mesocaecum was found to dilate venules and antagonized the vasoconstrictive effects of norepinephrine and promethazine [Bigazzi, M. et al., Acta Endocrinol. 112, 296-299 (1986); DelMese, A. et al., in Biology of Relaxin and its Role in the Human, Bigazzi, M. et al. (eds.) 291-293, Excerpta Medica, Amsterdam (1983)]. Chronic infusion of rat relaxin for two days to spontaneously hypertensive rats has reportedly led to blunted vasoconstrictive effects of norepinephrine and vasopressin on perfused mesenteric artery [Massicotte et al., Proc. Soc. Exp. Biol. Med. 190, 254-259 (1989)]. However, the physiological significance of these observations remains obscure, and it is not known whether or not relaxin affects total peripheral resistance.
Even more confusing and contradictory are some reports on the possible effects of relaxin on arterial blood pressure.
Miller et al. [J. Pharmacol. Exp. Ther. 120, 426-427 (1957)] first reported that injection of an extract of pregnant sow ovary caused a transient fall in blood pressure when injected into anesthetized dogs. In contrast, injection of an extract of up to 50 .mu.g purified porcine relaxin in anesthetized rats did not affect blood pressure [Porter et al., J, Endocrinol. 83, 183-192 (1979)].
Chronic infusion of rat relaxin has been reported to have no effect on arterial pressure in normotensive non-pregnant rats [St. Louis, J. and Massicotte, G., Life Sci, 37, 1351-1357 (1985)] or in pregnant rats [Ward, D.G., Am. J. Physiol. 261: in press (1991)].
Other publications indicate that intravenous injection of porcine relaxin in urethane anesthetized rats increases arterial pressure [Jones, S.A. and Summerlee, A.J.S., J. Physiol. (London), 381:37P (1986); Mumford, A.D. et al., J. Endocrinology 122., 747-755 (1989); Parry et al., J. Neuroendocrinology 253-58 (1990)]. The increase in arterial pressure is thought to be mediated predominantly by the release of vasopressin [Parry, L. J. et al., J. Neuroendocrinology, 2, 53-58 (1990)].
Intracerebroventricular injection of porcine relaxin was found to increase arterial pressure in the urethane anesthetized rat [Mumford, A.D., et al., J. Endocrinology 122, 747-755 (1989) ].
In view of these contradictory findings, the effect of relaxin on arterial pressure is at best unclear.
Intravenous or intracerebroventicular injection of porcine relaxin has been found to increase heart rate in the urethane anesthetized rat [Parry, L.J. et al., J. Neuroendocrinology 2, 53-58 (1990); Mumford, A. D. et al., J. Endocrinology 122, 747-755 (1989) ].
The interpretation of the various physiological effects of relaxin is very difficult, as little is known about the mechanism of action of relaxin at the molecular level. Relaxin is known to increase cAMP levels in uterine tissues or cells [Braddon, S.A. Endocrinology 102, 1292-1299 (1978); Sanborn, B. M. et al., Endocrinology 106, 1210-1215 (198)); Judson, D. G. et al., J. Endocrinology 87, 153-159 (1980); Chen, G. et al., Biol. Reprod. 39, 519-525; Kramer, S.M. et al., In Vitro Cell. Dev. Biol. 26 647-656 (1990)], and also in pituitary cells [Cronin, M. J. et al., Biochem. Biophys. Res. Commun. 148, 1246-1251 (1987)], but there is no conclusive evidence how cAMP acts as a mediator of relaxin actions.
Relaxin is reported to decrease uterine myosin light chain kinase activity and myosin light chain phosphorylation, which in turn inhibits the Ca.sup.++ -activated actomyosin ATPase in estrogen-primed rat uteri [Nishikori et al., Endocrinology 111, 1743-1745 (1982); Nishikori et al., J. Biol. Chem. 258, 2468-2474 (1983)]. The mechanism by which relaxin inhibits the myosin light chain kinase has yet to be elucidated.
Another important area of research for the understanding of the mechanism of action of relaxin, namely the relaxin receptors, is also in an early stage. Previously, Mercado-Simmen et al. [J. Biol. Chem. 255, 3617-3623 (1980); Endocrinology 110, 220-226 (1982); Biol. Reprod. 26, 120-128 (1982)] reported the partial characterization of rat and porcine uterine and porcine cervical relaxin receptors using .sup.125 I-labeled porcine relaxin. The .sup.125 I-labeled porcine relaxin was only partially purified over a Sephadex column, and was not substantially characterized chemically. The availability of biologically active, synthetic human relaxin whose structure is based on the nucleotide sequence obtained from ovarian cDNA clones, has recently allowed the first study of specific human relaxin receptors [Osheroff, P. L., J. Biol. Chem. 265, 9396-9401 (1990)].